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Background: Gli2, a transcription factor in the Hedgehog pathway, is overexpressed in castrate-resistant prostate cancer (PCa). Previously we showed that Gli2 overexpression increased transcriptional activity of androgen receptor (AR) and conferred androgen growth-independence to normally growth-dependent PCa cells. Here we localized the regions of AR-Gli2 protein interaction and determined the domains within Gli2 needed for AR co-activation., Methods: Co-immunoprecipitation and GST-pulldown assays were used to define AR-Gli binding domains. Co-activation assays using androgen-responsive promoter reporters were used to define Gli2 regions needed for AR co-activation. Chromatin immunoprecipitation (ChIP) assays were used to confirm nuclear interactions of Gli2 with AR in PCa cells., Results: The Gli2 C-terminal domain (CTD) is sufficient for AR co-activation. Two elements within the CTD were required: (1) an AR binding domain within aa628-897; and (2) at least part of the Gli2 transactivation domain within aa1252-1586. In turn, Gli2 binds the tau5/AF5 ligand-independent activation domain in the AR N-terminus. Mutations in the WxxLF motif in tau5/AF5 greatly diminished binding to Gli2-CTD. Gli2 interaction with AR tau5/AF5 was further substantiated by the ability of Gli2/Gli2-CTD to co-activate truncated AR splice variants (AR-V7/ARV567es). ChIP assays confirmed that Gli2 associates with chromatin at androgen response elements found near androgen-responsive genes in LNCaP cells. These assays also showed that AR associates with chromatin containing a Gli-response element near a Gli-responsive gene., Conclusion: Our findings indicate that Gli2 overexpression in PCa cells might support development of castration resistant PCa through AR co-activation and suggests that AR might modulate transcription from Gli2., (© 2014 Wiley Periodicals, Inc.)

Acquired intratumoral steroidogenesis is involved in progression of prostate cancer to castration resistant disease (CRPC) and a target for improved therapeutics. Recent work has shown that prostate cancer cells can acquire steroidogenic activity as they progress to a therapeutic-resistant state. However, benign prostate stromal cells (PrSCs) also have steroidogenic potential though they are often overlooked as a source of intratumoral androgens. Here, we present preliminary studies showing that the steroidogenic activity of primary human PrSCs is significantly increased by exposure to a Hedgehog agonist (SAG) or by transduction of PrSCs with lentiviruses that expresses active Gli2 (Gli2ΔN), a transcription factor that is triggered by Hh signaling. Comparative gene expression profiling on Chips, that was confirmed by quantitative real-time PCR, revealed that hedgehog agonist treatment induced in these cells expressions of hedgehog target genes (Gli1, Ptch1, and SCUBE1) plus a specific cadre of genes involved in cholesterol/steroid biosynthesis, metabolism, and transport. Genes involved downstream in steroid hormone generation, including CYP17A1 and CYP19A1 were also induced. Both the hedgehog agonist and the Gli2-expressing lentivirus significantly increased the output of testosterone (T) from PrSCs that were supplemented with dihydroepiandrosterone (DHEA), an adrenal precursor of T. Finally, knockdown of Gli2 by siRNA suppressed the ability of SAG to induce this response. Collectively, our data indicate that hedgehog/Gli signaling may be a factor in acquired intratumoral steroidogenesis of a prostate tumor through its actions on stromal cells in the tumor microenvironment and an influence for the development of CRPC., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Protocadherin-PC (PCDH-PC) expression is upregulated in apoptosis-resistant sublines of the LNCaP human prostate cancer (CaP) cell line. Here, we assess the role of PCDH-PC in CaP cells and its mRNA expression in human prostate tissues., Methods: LNCaP cells transfected with PCDH-PC were tested for their ability to grow in vitro and in vivo in androgen-deprived conditions. PCDH-PC mRNA expression was evaluated by semi-quantitative RT-PCR and by in situ hybridization., Results: PCDH-PC expression induced Wnt signaling in CaP cells and permitted androgen-independent growth of hormone-sensitive CaP cells. Expression of PCDH-PC-homologous transcripts was low and restricted to some epithelial cells in normal tissue and to CaP cells in tumors. However, hormone-resistant CaP cells expressed significantly higher levels of PCDH-PC-related mRNA., Conclusions: Our findings suggest a novel mechanism for the progression of CaP involving expression of PCDH-PC. This novel protocadherin induces Wnt signaling, promotes malignant behavior and hormone-resistance of CaP cells., (Copyright 2005 Wiley-Liss, Inc.)

Background: Androgen withdrawal, the standard therapeutic strategy for advanced prostate cancer, induces a rapid reduction of blood flow to prostate and prostate cancer tissues and the concomitant onset of a hypoxic environment in these tissues. To establish whether hypoxia-responsiveness (by means of the action of hypoxia inducible factor [HIF] -1alpha protein) might affect the tumorigenic or survival properties of prostate cancer cells, we studied how acute exposure of cultured human prostate cancer cells (LNCaP cell line) to hypoxia might alter their pattern of gene expression and their in vitro behavior., Methods: LNCaP cultures were placed in a hypoxia chamber for up to 24 hr and were compared with control (normoxic) cells for the expression of gene products by Western blotting and semiquantitative reverse transcription-polymerase chain reaction techniques., Results: Exposure of LNCaP cells to acute hypoxia activated a typical cellular hypoxia response characterized by up-regulation of HIF-1alpha and vascular endothelial growth factor protein expression. In contrast, expression of differentiation-specific proteins (prostate specific antigen and androgen receptor) or proliferative-regulatory proteins (c-myc, cyclin D1, p27) were down-regulated by hypoxia. Some of these latter changes (reduction of c-myc and cyclin D expression) were not accompanied by corresponding reduction of mRNAs and were abrogated by a proteosome inhibitor (MG132), suggesting that their loss was associated with their increased degradation rather than through transcriptional controls. The phosphorylation of Akt/protein kinase B and its downstream target, forkhead protein, was highly up-regulated by hypoxia, and cells exposed to transient periods of hypoxia became significantly less sensitive to an apoptotic stimulus (exposure to phorbol ester) when compared with normoxic cells., Conclusion: This study demonstrates that even acute hypoxia has the potential to drastically alter the growth, differentiation characteristics, and apoptotic sensitivity of a prostate cancer cell., (Copyright 2002 Wiley-Liss, Inc.)

Background: The protein encoded by the p53 gene is required for some forms of apoptosis and loss or mutations in this gene are found with increased frequency in advanced and hormone resistant human prostate cancers. In order to better appreciate whether reduction of wildtype p53 function in prostate cancer cells might contribute to the development of therapeutic-resistance by these cells, we created stable variants of the androgen-responsive, wild type p53-expressing human prostate cancer cell line, LNCaP, by transfection with expression vectors designed to reduce expression or function of wildtype p53 in them. These cells were then tested for their ability to form tumors in castrated male nude mice., Methods: A conditional eukaryotic expression vector (under tetracycline regulation) expressing antisense p53 cDNA was constructed and either directly transfected into LNCaP cells or tranduced into these cells using recombinant retroviruses containing the vector. Stably transfected/transduced cells (LNCaP/Asp53) were evaluated by Western blot analysis for the ability of doxycycline to reduce p53 protein expression and for their ability to form tumors in castrated male nude mice treated or untreated with doxycycline. Additionally, we derived an LNCaP subline (LNCaP/DD) stably expressing a dominant-negative form of p53 and tested these cells for their ability to form tumors in castrated male nude mice., Results: LNCaP/Asp53 cells showed reduced expression of p53 protein when cultured in a medium containing doxycycline and tested sublines were able to efficiently form tumors in castrated male nude mice only when the mice were treated with doxycycline. LNCaP/DD cells were readily able to form tumors in castrated male nude mice whereas parental LNCaP cells or control-transfected LNCaP cells were not., Conclusion: Loss of wildtype p53 function can contribute to the phenotype of hormone resistance of prostate cancer cells., (Copyright 2001 Wiley-Liss, Inc.)

Background: Earlier work from our laboratory highlighted the therapeutic potential of curcumin (turmeric), used as a dietary ingredient and as a natural anti-inflammatory agent in India and other Southeast Asian countries. This agent was shown to decrease the proliferative potential and induce the apoptosis potential of both androgen-dependent and androgen-independent prostate cancer cells in vitro, largely by modulating the apoptosis suppressor proteins and by interfering with the growth factor receptor signaling pathways as exemplified by the EGF-receptor. To extend these observations made in vitro and to study the efficacy of this potential anti-cancer agent in vivo, the growth of LNCaP cells as heterotopically implanted tumors in nude mice was followed., Methods: The androgen-dependent LNCaP prostate cancer cells were grown, mixed with Matrigel and injected subcutaneously into nude mice. Experimental group received a synthetic diet containing 2% curcumin for up to 6 weeks. At the end point, sections taken from the excised tumors were evaluated for pathology, cell proliferation, apoptosis, and vascularity., Results: Curcumin causes a marked decrease in the extent of cell proliferation as measured by the BrdU incorporation assay and a significant increase in the extent of apoptosis as measured by an in situ cell death assay. Moreover, a significant decrease in the microvessel density as measured by the CD31 antigen staining was also seen., Conclusions: Curcumin could be a potentially therapeutic anti-cancer agent, as it significantly inhibits prostate cancer growth, as exemplified by LNCaP in vivo, and has the potential to prevent the progression of this cancer to its hormone refractory state., (Copyright 2001 Wiley-Liss, Inc.)

Background: Promoter elements within the 5' DNA region of the rat C(3)1 gene have been shown to direct prostate-specific expression of gene products when they are fused through recombinant DNA procedures and used to produce transgenic mice. In order to test the in vivo effects of chronic overexpression of the mouse c-myc protooncogene on the prostate glands of transgenic mice, we created several lines of C(3)1-c-myc transgenic mice and then examined the phenotype of males with this genetic alteration., Methods: The modified promoter and 5' region of the rat C(3)1 gene was fused to the coding region of the mouse c-myc gene using recombinant DNA techniques. This DNA was used to create three different founder lines of transgenic mice. Tissues from males and females heterozygous for the transgene were examined for expression of the recombinant mouse c-myc mRNA by an RNase protection assay. Prostates from males were examined for expression of recombinant c-myc mRNA by in situ hybridization. Thin sections of fixed ventral prostates from males were analyzed by microscopy for histological abnormalities., Results: Three different lines of transgenic mice were obtained from these procedures. These mice demonstrated expression of recombinant mouse c-myc mRNA in the testis and ventral prostates of males and in the uterus of females. In situ hybridization demonstrated that the epithelial cells were the source of recombinant c-myc expression in the ventral prostates of the transgenic lines. Microscopic analysis of the ventral prostates from these mice demonstrated abnormalities in epithelial cell morphology seemingly typical of an intraepithelial neoplasia-like phenotype. However, none of the males of any of the lines developed overt prostatic adenocarcinoma over their lifetimes., Conclusions: Chronic overexpression of c-myc in the ventral prostate epithelial cells of C3(1)-c-myc transgenic mice leads to the development of epithelial cell abnormalities similar to those seen in low-grade prostatic intraepithelial neoplasia in humans. These abnormalities were not found to progress to adenocarcinoma over the lifetimes of the transgenic mice, suggesting the need for additional oncogenic changes in the pathway to prostatic adenocarcinomas. Furthermore, our cumulative experience with the use of the C3(1) gene promoter in the generation of transgenic mice suggests that the probasin promoter element provides a much more specific and effective means to target transgenes to the prostate glands of mice., (Copyright 2000 Wiley-Liss, Inc.)

Background: Blood flow to the rat ventral prostate gland is drastically reduced during the very early period after castration, and this reduction coincides with the appearance of striking degenerative changes within the prostatic vascular system. These early effects on the prostate vascular system are likely to be important for the subsequent regression of the ventral prostate that occurs in response to castration. Since the endothelial cells of the ventral prostate do not express androgen receptor protein (AR), we proposed that these early effects might be indirectly mediated by changes in the local expression of vascular regulatory factors. In order to evaluate whether vascular endothelial growth factor-A (VEGF-A) might be among the primary mediators of these effects, we measured expression of VEGF-A mRNA and protein in the rat ventral prostate gland prior to and within the first 3 days after castration., Methods: Ventral prostate tissues were obtained from control (unoperated) rats, sham-operated rats, or rats at sequential daily intervals (1-3 days) after castration. A quantitative RNase protection assay and a comparative RT-PCR assay were used to evaluate the extent to which the expression of VEGF-A mRNA in the ventral prostate was affected by castration. In situ immunohistochemistry, using an anti-VEGF-A antibody, was performed to localize VEGF-A protein in the various cells of the tissue. Western blot analysis and a quantitative ELISA assay using anti-VEGF-A antibodies were performed to determine how VEGF-A protein expression in the rat ventral prostate was affected by castration., Results: Results of VEGF-A mRNA analysis in the rat ventral prostate gland during the first 3 days after castration showed a biphasic change characterized by a transient reduction of VEGF-A mRNA expression (by approximately 50%) on the second day after castration that was restored to higher than control levels by the third day after castration. Immunohistochemical analysis for VEGF-A in control and castrated ventral prostates showed that the prostatic epithelial and smooth muscle cells were the major source of VEGF-A expression in this tissue. Quantitative analysis of VEGF-A protein expression by Western blot and ELISA methods confirmed a biphasic change in the expression of the polypeptide that correlated well with the results of the mRNA analyses., Conclusions: VEGF-A expression in the ventral prostate gland of the Sprague-Dawley rat is downregulated on the second day after castration but returns to control levels by the third day after castration. Since critical changes in the ventral prostate vascular system are already evident by 1 day after castration, we believe that these findings indicate that VEGF-A is not likely to be the critical or sole mediator of the early effects of castration on the vascular system of the rat ventral prostate gland., (Copyright 2000 Wiley-Liss, Inc.)

Background: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate., Methods: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF-beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy., Results: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals., Conclusions: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function., (Copyright 2000 Wiley-Liss, Inc.)

Background: Fas antigen/CD 95 is a 45-kDa transmembrane protein that can initiate intracellular signaling pathways, leading to apoptosis when it is clustered on the cell surface. A recent report claiming that the ventral prostate glands of lpr -/- mutant mice (lacking functional fas antigen) do not regress following castration prompted our analysis of the regressing rat ventral prostate gland for evidence that fas antigen might participate in the molecular process leading to prostate cell apoptosis after castration., Methods: An RNase protection assay and Western blotting analysis were used to quantify fas antigen mRNA and protein expression in the regressing rat ventral prostate gland. Immunoprecipitates of fas antigen from membrane preparations made from control or castrated rat prostates were analyzed for coprecipitation of FADD and RIP proteins to assess the activation state of the fas antigen before and after castration. Finally, prostate tissues obtained from two different strains of lpr -/- mutant mice were analyzed for induced apoptosis after castration by the TUNEL staining method., Results: Rat ventral prostate gland fas antigen mRNA and protein expression was upregulated approximately 3-5-fold in the 3-day castrated rat as compared to hormonally intact rats. Immunoprecipitates of fas antigen from membranes of ventral prostates from castrated rats contained significantly increased amounts of both FADD and RIP proteins when compared to those of intact or control operated rats. However, counts of TUNEL-labeled cells in the ventral prostate glands of castrated lpr -/- mice were not significantly different from those in castrated, genetically normal controls. Likewise, the morphology of apoptotic bodies formed in the prostates of castrated lpr -/- mice was indistinguishable from that in control animals., Conclusions: Fas antigen/CD-95, a protein that is involved in some forms of apoptosis, is upregulated during regression of the rat ventral prostate gland and becomes functionally "activated." However, our inability to distinguish any difference in the apoptosis rate or in the morphology of the apoptotic bodies formed in response to castration between lpr -/- mice and genetically normal controls indicates that, contrary to the prior report, functional fas protein is not required for castration-induced prostate cell apoptosis.

Background: There is growing interest as to the potential role of the prostatic vascular system in mediating the effects of androgenic steroids on growth control of the prostate. Here we describe the use of a vascular corrosion casting technique that enables the visual characterization of the vascular system of the rat ventral prostate and the description of some unique morphological features of the mature rat prostate gland that were never previously observed., Methods: Anesthetized adult male Sprague-Dawley rats were vascularly perfused with a fixative solution and then with a catalyzed methacrylate-based casting solution that was allowed to polymerize in situ. Ventral prostate glands were dissected from these rats and the tissues were subsequently dissolved in a corrosive solution, leaving residual vascular casts. The casts were then examined by scanning electron microscopy for pertinent morphological features., Results: Vascular corrosion casts of individual lobes of the mature rat ventral prostate revealed a complex vascular structure that entered into the prostate near the base of the bladder. The morphological correspondence of the prostatic vasculature to the previously described ductal organization of the prostate was readily apparent. Examination of the entire vascular complex (under low-power scanning electron microscopy) revealed three differing surface features of the tissue and suggested that the glandular elements of the ventral prostate were directionally oriented towards a single surface (the anterior-lateral surface). An opposing face (posterior-medial) of the tissue demonstrated some unique spiral vessels, suggesting the need for a potential stretch-compensation mechanism at the prostate surface immediately adjacent to the bladder., Conclusions: Vascular casts of the rat ventral prostate gland reveal the obvious ductal organization of the prostatic parenchyma and demonstrate that these ducts are directionally oriented so that the glands of the prostate (at the distal tips of the ducts) uniformly lie near the anterior-lateral surface of the tissue. Unusual spiral arterioles found proximal to the bladder surface suggest that the rat ventral prostate gland has acquired the means to adapt to an anatomical position adjacent to a tissue (bladder) that acutely varies in size during the micturition cycle.

Background: Androgenic steroids regulate the development and size of the mammalian prostate gland. The mechanism(s) for this growth control might involve a direct effect on prostate cell proliferation and survival as well as more complex effects on the tissue environment supporting nourishment and oxygenation. In this study, we evaluated an animal model of androgen action on the prostate, the rat ventral prostate gland, to determine whether acute androgen withdrawal, by means of castration, might alter the primary blood flow to the prostate gland and for the effects of castration on prostatic endothelial cell viability., Methods: Groups of rats studied included intact control males, males that had been surgically castrated, or males that received a sham-surgical castration. Relative blood flow (RBF) to the rat ventral prostate glands and rat bladders were measured at 18 and 24 hr after castration or sham castration using a fluorescent microsphere infusion technique. Thin sections from fixed and embedded rat ventral prostate glands obtained from unoperated or 12-hr castrated rats were analyzed by the TUNEL immunostaining technique to microscopically identify and quantify apoptotic epithelial, stromal, and endothelial cells., Results: RBF to the rat ventral prostate was reduced by 38%, at 18 hr after castration when compared with intact or sham-operated rats and by 45% at 24 hr after castration (P=0.038 unoperated/0.025 sham operated). In contrast, RBF to the bladder was not significantly different between any of the groups in the 24-hr castrate experiment. TUNEL staining analysis of ventral prostate tissues obtained from 12-hr castrated rats showed only rare TUNEL-positive epithelial cells similar to the control tissue but significantly increased TUNEL labeling for endothelial and other ventral prostate stromal cells., Conclusions: Castration resulted in a rapid and significant reduction of blood flow to the mature rat ventral prostate gland that was not seen in the bladder. This reduction precedes the appearance of apoptosis in the epithelial cells of the tissue but more coincided with the appearance of TUNEL-positive prostate vascular endothelial and stromal cells, suggesting that androgens support the survival of cells in the vascular and stromal compartment of the rat prostate as well as in the prostatic epithelium. These preliminary data support the concept that androgen action on the prostate might involve primary regulation of prostate blood flow and prostate vascular cell vitality.

Background: The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo., Methods: A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis., Results: The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells., Conclusions: This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.

Background: Recent hypotheses to explain the etiology of abnormal growth associated with prostate disease have invoked perturbations in the rate of apoptosis as an important contributor to the onset and progression of these diseases. For this reason, the apoptosis suppressing oncoprotein bcl-2 has come under scrutiny with regards to its role in prostate diseases. In order to evaluate the role of bcl-2 in human prostate disease and to develop an animal model to test anti-bcl-2 therapies, we generated transgenic mice in which bcl-2 expression is targeted to the mouse prostate gland., Methods: Mouse embryos were microinjected with recombinant DNA constructed by fusing a modified rat C3(1) promotor element to cDNA encoding human bcl-2. Presence of the C3(1)-bcl-2 transgene in progeny was identified by Southern blot and polymerase chain reaction (PCR) analysis. RNase protection assays were used to analyze RNA from 15 organs of these mice. Western blot assays and immunohistochemical staining were used to confirm the tissue-specific protein expression of human bcl-2 and its cellular localization., Results: Three lines of C3(1)-bcl-2 transgenic mice were established. Founder mice carried 2-20 copies of the transgene. Expression of human bcl-2 from the transgene was limited to the prostate gland and testis of males as well as the uterus of females. In the prostate gland, human bcl-2 protein was found only in prostatic epithelial cells. Microscopic analysis of prostate glands from individual males (three lines) showed that these glands were often abnormal, with increased accumulation of cells in the prostatic stroma as well as the epithelium., Conclusions: These transgenic mice appear to provide a novel animal model for studying neoplastic development of the prostate, with particular emphasis on the bcl-2 protein and the role of apoptosis regulation in such development.

Androgen deprivation subsequent to castration of an adult male rat results in the regression of sexual accessory tissues. Regression of these tissues involves the massive death of androgen-dependent cells. Using the rat ventral prostate gland as a model to study androgen-programed cell death, we have characterized a series of molecular events that accompany its regression. This analysis has shown that there was a sequential induction of specific gene transcripts in the ventral prostate gland following castration. The first event in this cascade was an abrupt induction of transcripts encoding c-fos. Since c-fos expression has been linked to perturbations in intracellular Ca2+ levels, we investigated whether membrane-mediated Ca2+ flux might be an early physiological step involved in the death of prostatic cells. To test this, rats were treated simultaneously upon castration with either verapamil or nifedipine, two different calcium channel antagonist drugs. Compared to the ventral prostate glands of untreated castrated rats, the glands of the calcium channel antagonist-treated rats showed a significant delay in all parameters associated with regression (loss of wet weight and DNA content and delay in histological changes associated with prostatic regression). The seminal vesicle glands of treated rats also showed signs of delayed regression. Furthermore, calcium channel antagonists suppressed the induction of transcripts encoding both c-fos and testosterone-repressed prostate message-2 (TRPM-2), a gene expressed exclusively by dying cells, during the first 62 hr following castration. These findings further support a role for calcium ion influx in the pathway leading to hormonally programed prostate cell death, and suggest the intriguing possibility that modulation of this activity can alter the process by which cells die.

We examined a series of 29 surgical specimens of benign and malignant human prostate tissue for the expression of both the cHa-ras and c-myc protooncogenes. Northern blots were prepared using polyadenylated mRNA extracted from nine prostatic adenocarcinomas, 19 benign hypertrophied prostates (BPH) and one normal prostate. When the Northern blots were hybridized to a probe for cHa-ras, only one specimen of BPH showed an appreciable amount of the 1.2-kb transcript homologous to cHa-ras. Upon reprobing these blots with c-myc, six cancers showed a considerable amount of a 2.4-kb transcript homologous to c-myc. Three other cancers and all the benign tissue showed little or no detectable 2.4-kb c-myc transcript. On retrospective analysis, the cancers with elevated c-myc transcripts were found to have a Gleason score of 5 and above (poorly differentiated tumors), while the cancers with little or no c-myc transcripts were all of Gleason score 4 and lower. Finally, we compared our ability to detect c-myc transcripts in mRNA extracted from a surgically derived prostate tumor with mRNA extracted from the same tumor subject to a sham electrocautery procedure, as would occur during transurethral resection. The electrocautery procedure decreased both the intensity and the integrity of the c-myc signal in mRNA from the tumor. Thus, our exclusive use of surgically derived prostate tumors may be the reason we are able to detect an elevation in the expression of c-myc mRNA in high-grade tumors.